PROJECT SUMMARY: Asparagine (N) linked glycosylation (NLG) is the most common protein modification of membrane and secretory proteins in eukaryotes. However, A major challenge in glycobiology has been to identify and understand the cellular mechanisms and non-essential gene products that regulate NLG. Furthermore, though this pathway is target rich and involves at least 34 gene products, pharmacologic inhibitors that regulate this process have not been available. We have designed and implemented a bioluminescent imaging strategy to detect changes in NLG site occupancy at the cellular level. This platform can be integrated in experiments that range from microplate detection to in vivo imaging, and high throughput screening efforts have delivered two structurally independent classes of NLG inhibitors. The first inhibitor, NGI-1, blocks oligosaccharyltransferase activity and causes loss of fidelity for this multi-subunit enzyme complex. We plan to identify the specific amino acids and NGI-1 binding pocket in order to more precisely understand the mechanism of small molecule OST inhibition. The cellular target for the second inhibitor has yet to be defined and we therefore propose methodologies to investigate and identify this novel mechanism of action for NLG inhibition. We have also designed and validated a fluorescent imaging approach which detects abnormal glycosylation and that can be used to detect heterogeneity of glycan site occupancy at the single cell level. We propose to advance this technique to both in vitro and in vivo models in order to both investigate genetic factors that regulate NLG and to examine glycosylation differences in mouse organs and tissues. Together this work will provide new insights into the regulation of NLG by genetic factors or small molecule inhibitors, and provide a technique for quantifying dynamic changes and the heterogeneity of NLG in specific cells both in vitro and in vivo.